Plasma leptin, glucose and non-esterified fatty acid variations in dromedary camels exposed to prolonged periods of underfeeding or dehydration.

The involvement of plasma leptin in the adaptation of dromedary camels to harsh conditions such as food or water shortages was studied through 2 experiments. In experiment 1, fourteen female camels were either fed at 68% of maintenance energy requirements (MER) during 112d (n=4) or overfed at 134% of MER during the first 56d and then underfed at 17% of MER the next 56d (OV-UN, n=5), or underfed and then overfed for the same durations and energy intake levels (UN-OV, n=5). Weekly plasma samples showed that leptin, glucose and non-esterified fatty acid (NEFA) concentrations were significantly modulated by energy intake level. NEFA increased sharply but transiently in underfed camels of the UN-OV or OV-UN groups, whereas glucose and leptin concentrations decreased with underfeeding and increased with overfeeding with more significant effects in camels that were previously overfed or underfed, respectively. In experiment 2 twelve female camels were either normally watered (n=6) or dehydrated (n=6) during 23d and then rehydrated during 4d. Dehydration specifically increased blood hematocrit, plasma NEFA and glucose whereas leptin decreased slightly. For both experiments, leptinemia was positively related to hump adipocyte volume. Taken together these results provide new data for a better understanding of lipid and energy metabolism in camels.

Development of a monoclonal antibody-based immunoassay for specific quantification of bovine milk alkaline phosphatase.

The detection of alkaline phosphatase (ALP) activity is used as a legal test to determine whether milk has been adequately pasteurized or recontaminated with raw milk. However, a wide variety of microorganisms produce both heat labile and heat stable ALPs which cannot be differentiated from the milk ALP by current enzymatic methods. Monoclonal antibodies specific of the bovine milk ALP were obtained in mice from a raw bovine milk ALP preparation. Coated in microtitre plates, these antibodies specifically capture the bovine milk ALP from dairy products. After washing, the enzymatic activity of the captured ALP is revealed by adding p-nitrophenyl-phosphate as a substrate. This simple immunoassay does not react with ALPs of intestinal or bacterial origin and, once optimized, was found to be the first immunoassay suitable to detect raw milk in boiled milk down to a 0.02% dilution. Moreover, in contrast with competitive indirect ELISA formats, the capture immunoassay does not require purified ALP.

Inactivation-denaturation kinetics of bovine milk alkaline phosphatase during mild heating as determined by using a monoclonal antibody-based immunoassay.

A monoclonal antibody based capture immunoassay has been recently developed for the specific quantitation of bovine milk alkaline phosphatase (ALP) without interference by contaminating microbial or fungal ALPs (Geneix et al. 2007). This immunoassay was used to study the kinetics of ALP heat denaturation in bovine milk over a range 50-60 degrees C for 5 to 60 min using a colorimetric quantification of the enzyme activity as a reference test. A denaturation midpoint was obtained at 56 degrees C for a 30 min heating. Thermal inactivation was found to follow first order kinetics and is characterized by z value of 6.7 deg C (D60 degrees C=24.6 min) and 6.8 (D60 degrees C=23.0 min) for respectively immunoassay and colorimetric assay. The high values of enthalpy of activation and the positive values of the entropy of activation and free energy of activation indicate that during denaturation ALP underwent a large change in conformation. The results of the immunoassay were highly correlated (r=0.994) with those obtained by the colorimetric assay. A similar high correlation (r=0.998) was obtained when industrially thermized milks (62-67 degrees C for 20-90 s) were analysed by both techniques. These results indicated that 1) thermally induced epitopic structural changes recognized by the capture monoclonal antibody are concomitant with or occur after the loss of enzymatic activity and 2) quantification of ALP by the specific immunoassay is appropriate for determining mild time/temperature treatment of milk and for the control of milk pasteurization.

Camel (Camelus dromedarius) immunoglobulin G, alpha-lactalbumin, serum albumin and lactoferrin in colostrum and milk during the early post partum period.

Colostrum and milk samples from twelve Tunisian camels were analysed for concentration of immunoglobulin G (IgG), alpha-lactalbumin (alpha-la), serum albumin (CSA) and lactoferrin throughout the first 14 milkings post partum (7 days of lactation) using single radial immunodiffusion assay. Concentrations (mg/ml, means+/-SD) at first milking were IgG, 100.7+/-60.4; alpha-la, 2.2+/-0.7; CSA, 8.5+/-3.6 and lactoferrin, 1.2+/-0.3. Large variations were recorded for IgG and CSA concentrations (11.8-211.1 mg/ml and 2.9-13.8 mg/ml respectively) Concentrations of IgG and CSA dropped abruptly in the subsequent milkings while alpha-la concentration increased until milking 5 and then decreased slowly. Lactoferrin dropped only from milking 7. Mean IgG concentrations were 3.6 and 2.5 mg/ml at milking 9 and 13 respectively. However, IgG concentration did not differ significantly, at the 1% level, from milkings 11 to 14. The contribution of CSA to the increase in whey proteins in early milks was greater than that described in the bovine and caprine species.

Immunochemical quantification of heat denaturation of camel ( Camelus dromedarius ) whey proteins.

The major whey proteins IgG, serum albumin and alpha-lactalbumin were purified from camel milk using gel permeation and ion-exchange chromatography. Specific antisera against each of them were raised and used to quantify their heat denaturation in early or mature milk over a range of 60-90 degrees C for 10-60 min using the single radial immunodiffusion technique. The heat denaturation midpoints for the mature milk heated 30 min were 67.2, 73.0 and 77.5 degrees C for IgG, albumin and alpha-lactalbumin respectively. The early milk was more sensitive to heat treatment with coagulation at low temperature and heat denaturation midpoints of 64.8, 71.6 and 72.6 degrees C respectively. This difference was related to the high IgG content of the early milk (12.6 mg/ml v. 0.5 mg/ml for the mature milk) and stresses the importance of monitoring the IgG level of milk to assess the absence of colostrum.

Differentiation of gelatins using polyclonal antibodies raised against tyrosylated bovine and porcine gelatins.

Gelatin is obtained from bones and hides/skin, mainly from cows and pigs using alkaline or acidic processes. The use of bovine gelatin in feed, food, and pharmaceutical products has been restricted by regulatory authorities as a consequence of the outbreak of bovine spongiform encephalopathy (BSE). On the other hand, some religions ban the porcine gelatin for human consumption. Thus, there is a need for methods able to control the species origin of gelatins. The large similarity in structure of gelatins from different origins has made unsuccessful their differentiation by physicochemical methods. Moreover, the development of immunochemical methods has been hampered by the poor immunogenicity of gelatins. We obtained high titers antibodies upon immunization of rabbits with tyrosylated bovine and porcine gelatins. Using indirect and competitive indirect ELISAs we observed large differences in titers and specificity among rabbits and during the course of immunization. Some of the antisera were not sensitive to the species origin of raw material or to the process used for gelatin production and could be used for gelatin quantitation in food. Other antisera detected the porcine acidic gelatins with 10- to 30-fold higher sensitivity than their bovine counterparts and could be used for the differentiation of the species origin of gelatins. Lastly, other antisera were highly sensitive to subtle changes in conformation of gelatins obtained by alkaline or acidic processes such as a 1,000-fold higher reactivity of bovine acid hide gelatin compared to that of its limed counterpart or a 30,000-fold higher reactivity of porcine acid bone compared to that of its limed counterpart; such antisera could be used to monitor the process induced structural changes of collagen during its transformation to gelatin.

Front-face fluorescence spectroscopy allows the characterization of mild heat treatments applied to milk. Relations with the denaturation of milk proteins.

Emission and excitation spectra of intrinsic fluorophores present in milk were used to evaluate changes in milk following thermal treatments in the 57-72 degrees C temperature range from 0.5 min up to 30 min. Alternatively, the concentrations of native alkaline phosphatase, lactoferrin, immunoglobulin G, bovine serum albumin, beta-lactoglobulin, and alpha-lactalbumin were determined in the same samples by enzymatic and immunochemical techniques. As principal component analysis applied to the normalized fluorescence spectra successfully discriminated different milk samples according to the temperature and time of thermal treatment, principal component regression was applied to predict the amounts of the native proteins investigated using fluorescence data. The results showed strong correlations between measured and predicted data for alkaline phosphatase and beta-lactoglobulin. This study has demonstrated that front-face fluorescence spectroscopy has a promising potential to become a rapid and nondestructive analytical technique for the evaluation of physicochemical changes in milk induced by low thermal treatment.

Caprine immunoglobulin G, beta-lactoglobulin, alpha-lactalbumin and serum albumin in colostrum and milk during the early post partum period.

Colostrum and milk samples from 20 goats were analysed for concentrations of immunoglobulin G (IgG), beta-lactoglobulin (beta-lg), alpha-lactalbumin (alpha-la) and serum albumin (CSA) throughout the first 14 milkings post partum (7 d of lactation) using single radial immunodiffusion assay. Concentrations (mg/ml, means +/- SD) at first milking were IgG 47.9 +/- 25.5, beta-lg 30.7 +/- 10.4, alpha-la 2.77 +/- 0.82 and CSA 2.97 +/- 2.46 mg/ml. Large variations were recorded for IgG concentrations (19.9-94.5 mg/ml) and beta-lg (9.3-49.8 mg/ml). Concentrations of IgG, beta-lg and CSA dropped abruptly in the subsequent milkings and alpha-la concentration decreased slowly. Mean IgG concentration was < 2 mg/ml after 7 milkings and < 1 mg/ml after 11 milkings. However, IgG concentration does not differ significantly, at the 1% level, from milkings 7-14. The contribution of beta-lg to the increase in whey proteins in early milks was greater than that of IgG from milkings 5 to 14. The results were tabulated to make it possible to calculate the excess of whey proteins that would be obtained if early milks were illegally added to milk supply.

Characterisation of Ag1, the major species-specific contaminant of bovine crude heparin, and its identification as an aprotinin/heparin complex.

Heparin is a potent anticoagulant polysaccharide purified for decades from ruminants or porcine tissues. However, with the emergence of bovine spongiform encephalopathy (BSE), the source of pharmaceutical heparin is currently restricted to porcine intestinal mucosa. A major species-specific contaminant, called Ag1, has recently been identified in bovine crude heparin [Rivera et al., J. Pharm. Biomed. Anal., submitted] and used to develop an enzyme-linked immunosorbent assay (ELISA) for the species origin control of crude heparins [Levieux et al., J. Immunoassay, submitted]. In this report, we describe the different investigations, which were carried out to identify Ag1. This antigen was first localised by immunohistological studies essentially in the connective tissue of the bovine small intestine. After extraction from an intestinal extract by immuno-affinity chromatography, Ag1 was isolated as a single band by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Ag1 was then partly sequenced and identified as an aprotinin/heparin complex. Aprotinin, also known as the bovine pancreatic trypsin inhibitor (BPTI), is present with heparin in mast cells, and is very resistant to heat, pH, chemical treatments and proteolytic digestion. The stability of Ag1 towards the different treatments performed during heparin extraction process allows this protein to remain in sufficient amounts in crude heparin and makes it an ideal target for the immunochemical control of the absence of bovine material in crude heparins.

Development and evaluation of a sandwich ELISA for quantification of the 20S proteasome in human plasma.

Because quantification of the 20S proteasome by functional activity measurements is difficult and inaccurate, we have developed an indirect sandwich enzyme-linked immunosorbent assays (ELISA) for quantification of the 20S proteasome in human plasma. This sandwich ELISA uses a combination of a monoclonal antibody (mcp 20) recognizing the C2-beta subunit of human 20S proteasome (Mr approximately 30,000) and a polyclonal rabbit anti-20S antibody which labels different subunits of the complex. The detection limit of the assay was established as 10 ng/ml (n=10, mean of zero standard+2 S.D.) and the recovery rate ranged from 96% to 104%. The within-run and between-run coefficients of variation (CV) ranges were 2.8-3.3 and 3.0-3.4, respectively. Using serial dilutions of plasma to which various amounts of purified 20S proteasome were added, a linear dose-response was observed between 102 and 2050 ng/ml with a slope of 1.004 and a coefficient of determination r(2) of 0.99. In a preliminary experiment performed on a limited number of patients, the present assay was used to quantify the 20S proteasome in plasma from healthy subjects (n=11) and from a limited number of patients with various diseases (two patients with each of the following diagnoses: acute myeloid leukaemia, chronic myeloproliferative syndromes, Hodgkin's disease and solid tumors). The average concentration of 20S proteasome in plasma from normal subjects was found to be 2319+/-237 ng/ml (n=11). With reference to this normal range, the plasma proteasome concentration was found to be increased in most of these pathological state and as high as 1200% when solid tumors had been detected. For patients with Hodgkin's disease, the changes were more variable whereas in patients with chronic lymphocytic leukaemia, the proteasome concentration was raised during the acute phase of disease and decreased during therapy. We suggest that this robust, accurate and highly reproducible assay could be used to quantify proteasome in human plasma and investigate its value as a biological marker for various malignant and nonmalignant diseases.